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In vitro Selection and Interaction Studies of a DNA Aptamer Targeting Protein A.

Stoltenburg R, Schubert T, Strehlitz B - PLoS ONE (2015)

Bottom Line: Structural investigations and sequence truncation experiments of the selected aptamer for Protein A led to the conclusion, that a stem-loop structure at its 5'-end including the 5'-primer binding site is essential for aptamer-target binding.Cross specificity to other proteins was not found.The application of the aptamer is directed to Protein A detection or affinity purification.

View Article: PubMed Central - PubMed

Affiliation: UFZ-Helmholtz Centre for Environmental Research, Department of Soil Ecology, Halle, Germany.

ABSTRACT
A new DNA aptamer targeting Protein A is presented. The aptamer was selected by use of the FluMag-SELEX procedure. The SELEX technology (Systematic Evolution of Ligands by EXponential enrichment) is widely applied as an in vitro selection and amplification method to generate target-specific aptamers and exists in various modified variants. FluMag-SELEX is one of them and is characterized by the use of magnetic beads for target immobilization and fluorescently labeled oligonucleotides for monitoring the aptamer selection progress. Structural investigations and sequence truncation experiments of the selected aptamer for Protein A led to the conclusion, that a stem-loop structure at its 5'-end including the 5'-primer binding site is essential for aptamer-target binding. Extensive interaction analyses between aptamer and Protein A were performed by methods like surface plasmon resonance, MicroScale Thermophoresis and bead-based binding assays using fluorescence measurements. The binding of the aptamer to its target was thus investigated in assays with immobilization of one of the binding partners each, and with both binding partners in solution. Affinity constants were determined in the low micromolar to submicromolar range, increasing to the nanomolar range under the assumption of avidity. Protein A provides more than one binding site for the aptamer, which may overlap with the known binding sites for immunoglobulins. The aptamer binds specifically to both native and recombinant Protein A, but not to other immunoglobulin-binding proteins like Protein G and L. Cross specificity to other proteins was not found. The application of the aptamer is directed to Protein A detection or affinity purification. Moreover, whole cells of Staphylococcus aureus, presenting Protein A on the cell surface, could also be bound by the aptamer.

No MeSH data available.


Related in: MedlinePlus

Most abundant aptamer sequences in the selected aptamer pool.Seven groups with 3–8 homologous sequences (#: number of homologous sequences) were identified among the sequenced aptamer clones. The representative aptamer clone of each group is shown. The specific primer binding sites at the 5’- and 3’-end of the aptamer clones are colored in red and blue, respectively.
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pone.0134403.g001: Most abundant aptamer sequences in the selected aptamer pool.Seven groups with 3–8 homologous sequences (#: number of homologous sequences) were identified among the sequenced aptamer clones. The representative aptamer clone of each group is shown. The specific primer binding sites at the 5’- and 3’-end of the aptamer clones are colored in red and blue, respectively.

Mentions: The selection of DNA aptamers for Protein A as a cell surface protein of Staphylococcus aureus was performed using the FluMag-SELEX procedure [23]. This SELEX variant is based on magnetic beads as immobilization matrix for the target molecules (herein Protein A/Strep-MB) and on the utilization of a fluorescence label for quantification of the DNA during the SELEX process (herein 5’-fluorescein modified oligonucleotides) (S1 Fig). After 7 SELEX rounds, a stepwise enrichment of target-bound oligonucleotides was observed for the next 4 selection rounds (S2 Fig). The selection conditions were changed from round 7 onwards concerning the insertion of a negative selection step with unmodified Strep-MB, the elution step, and the stringency of washing steps of the binding complexes in round 9–11 (S2 Fig). The selected aptamer pool from SELEX round 11 was cloned and 88 individual aptamer clones were sequenced and further characterized. Seven sequence groups could be identified containing 3–8 homologous aptamer clones (Fig 1). Five sequences of the 88 individual aptamer clones were present twice in the pool (S3 Fig), but the majority of clones were orphans [30]. This indicates that the SELEX process results in a very heterogeneous aptamer pool.


In vitro Selection and Interaction Studies of a DNA Aptamer Targeting Protein A.

Stoltenburg R, Schubert T, Strehlitz B - PLoS ONE (2015)

Most abundant aptamer sequences in the selected aptamer pool.Seven groups with 3–8 homologous sequences (#: number of homologous sequences) were identified among the sequenced aptamer clones. The representative aptamer clone of each group is shown. The specific primer binding sites at the 5’- and 3’-end of the aptamer clones are colored in red and blue, respectively.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4519192&req=5

pone.0134403.g001: Most abundant aptamer sequences in the selected aptamer pool.Seven groups with 3–8 homologous sequences (#: number of homologous sequences) were identified among the sequenced aptamer clones. The representative aptamer clone of each group is shown. The specific primer binding sites at the 5’- and 3’-end of the aptamer clones are colored in red and blue, respectively.
Mentions: The selection of DNA aptamers for Protein A as a cell surface protein of Staphylococcus aureus was performed using the FluMag-SELEX procedure [23]. This SELEX variant is based on magnetic beads as immobilization matrix for the target molecules (herein Protein A/Strep-MB) and on the utilization of a fluorescence label for quantification of the DNA during the SELEX process (herein 5’-fluorescein modified oligonucleotides) (S1 Fig). After 7 SELEX rounds, a stepwise enrichment of target-bound oligonucleotides was observed for the next 4 selection rounds (S2 Fig). The selection conditions were changed from round 7 onwards concerning the insertion of a negative selection step with unmodified Strep-MB, the elution step, and the stringency of washing steps of the binding complexes in round 9–11 (S2 Fig). The selected aptamer pool from SELEX round 11 was cloned and 88 individual aptamer clones were sequenced and further characterized. Seven sequence groups could be identified containing 3–8 homologous aptamer clones (Fig 1). Five sequences of the 88 individual aptamer clones were present twice in the pool (S3 Fig), but the majority of clones were orphans [30]. This indicates that the SELEX process results in a very heterogeneous aptamer pool.

Bottom Line: Structural investigations and sequence truncation experiments of the selected aptamer for Protein A led to the conclusion, that a stem-loop structure at its 5'-end including the 5'-primer binding site is essential for aptamer-target binding.Cross specificity to other proteins was not found.The application of the aptamer is directed to Protein A detection or affinity purification.

View Article: PubMed Central - PubMed

Affiliation: UFZ-Helmholtz Centre for Environmental Research, Department of Soil Ecology, Halle, Germany.

ABSTRACT
A new DNA aptamer targeting Protein A is presented. The aptamer was selected by use of the FluMag-SELEX procedure. The SELEX technology (Systematic Evolution of Ligands by EXponential enrichment) is widely applied as an in vitro selection and amplification method to generate target-specific aptamers and exists in various modified variants. FluMag-SELEX is one of them and is characterized by the use of magnetic beads for target immobilization and fluorescently labeled oligonucleotides for monitoring the aptamer selection progress. Structural investigations and sequence truncation experiments of the selected aptamer for Protein A led to the conclusion, that a stem-loop structure at its 5'-end including the 5'-primer binding site is essential for aptamer-target binding. Extensive interaction analyses between aptamer and Protein A were performed by methods like surface plasmon resonance, MicroScale Thermophoresis and bead-based binding assays using fluorescence measurements. The binding of the aptamer to its target was thus investigated in assays with immobilization of one of the binding partners each, and with both binding partners in solution. Affinity constants were determined in the low micromolar to submicromolar range, increasing to the nanomolar range under the assumption of avidity. Protein A provides more than one binding site for the aptamer, which may overlap with the known binding sites for immunoglobulins. The aptamer binds specifically to both native and recombinant Protein A, but not to other immunoglobulin-binding proteins like Protein G and L. Cross specificity to other proteins was not found. The application of the aptamer is directed to Protein A detection or affinity purification. Moreover, whole cells of Staphylococcus aureus, presenting Protein A on the cell surface, could also be bound by the aptamer.

No MeSH data available.


Related in: MedlinePlus